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    • 专利摘要:
    The invention relates to a method for producing tolerogenic dendritic cells (tolDCs) with specific antigens, comprising the steps of: (a) culturing precursors of dendritic cells in an an animal-serum-free medium, using cytokines, IL-4 and GM-CSF, in order to differentiate same in dendritic cells; (b) producing apoptotic cells; (c) culturing the dendritic cells obtained in step (b) in the presence of compounds having anti-inflammatory activity; (d) co-culturing the dendritic cells from step (d) with the apoptotic cells from step (c), such as to stimulate the endocytosis of the apoptotic cells by the dendritic cells; (e) and, by means of identification based on phenotypic evaluation, determining the production of tolerogenic dendritic cells (tolDCs) with specific antigens. The invention also relates to the tolDC cells produced with said method and to the use of said tolDCs with specific antigens in the production of a drug suitable for the treatment of systemic lupus erythematosus.
    公开号:ES2609068A2
    申请号:ES201690066
    申请日:2015-06-05
    公开日:2017-04-18
    发明作者:Carolina LLANOS MUÑOZ；Alexis Mikes Kalergis Parra；Fabian Alejandro VEGA TAPIA；Andy Igor TORRES BAEZA
    申请人:Pontificia Universidad Catolica de Chile；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION The present invention relates to the production of tolerogenic dendritic cells for specific antigens designed for use in autoimmune diseases, in particular for the treatment of systemic lupus erythematosus (SLE).
    STATE OF ART Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease of unknown etiology that primarily affects women of childbearing age with a prevalence of 124 cases per 100,000 people. From a clinical point of view, lupus affects multiple organs, including the kidneys, heart, lungs, skin and musculoskeletal and hematological systems, among others. It also presents an unpredictable clinical evolution characterized by reactivations or outbreaks alternated with periods of remission. Although the mortality of SLE has decreased significantly in the last 50 years, a patient who develops SLE at the age of 20 has a 15% chance of dying at 35 years of age from lupus itself or an associated complication. The SLE also has an important aggregate morbidity. As an example, one of the most relevant complications in terms of the impact on survival and quality of life for patients is lupus nephritis (NL) that affects up to 50% of patients with lupus. Despite the important efforts made to optimize the treatment of this manifestation, still 10-30% of patients presenting with NL reach terminal renal failure and therefore require dialysis and / or renal transplantation. Also, patients with lupus have a mortality between 2 and 5 times higher than the general population,

    they present up to 23% of work disability and in the case of women of reproductive age, this is accompanied by difficulties in conceiving and greater morbidity-mortality during pregnancy than healthy women. Likewise, the FDA approved the year 2011 for the first time in 30 years a new drug (belimumab) for the treatment of SLE. However, this medicine and the rest of the drugs commonly used in this disease, including mycophenolate mofetil (MMF), cyclophosphamide and steroids, are non-curative and non-specific immunosuppressive treatments that are associated with important complications such as opportunistic infections. Recently, the research group of the ALMS project (NCT00377637) that studied the use of MMF in lupus nephritis, published the results of the maintenance phase of said trial where the drug under study v / s azathioprine was compared. The results show that 33.3% of the patients who used azathioprine and 23.5% of those who used MMF had serious adverse events that led to the withdrawal of the study in 39.6% and 25.2% of the patients. patients respectively. Other biological agents approved for use in other autoimmune pathologies such as rituximab, have not demonstrated efficacy in controlled clinical studies in lupus and, like traditional immunosuppressants, may also contribute to the onset of opportunistic infections. The present invention consists of a method for obtaining autologous autologous dendritic cells (DCs) specific for apoptotic cells for immunotherapy for SLE. In the search for the state of the art, documents related to obtaining and using dendritic cells for the treatment of immunological diseases were found. The closest document to the technology under study is patent application WO2001085207 where they propose to use Apoptotic Cells to decrease the ability of DCs to stimulate a cellular response through induction of anergy to T cells. In addition, they include various alternatives to modulate the presentation of apoptotic antigens by DCs and the use of maturing factors of DCs. In this case, it is the apoptotic cells that exert the modulating action on the function of the cells

    dendritic Nowhere is the relationship of apoptotic cells with Systemic Lupus Erythematosus mentioned. In the present invention, the objective of the use of apoptotic cells is different, it does not seek to be per se what promotes the modulation of the function of the DCs and it is not relevant how the apoptotic antigens are presented by the DCs that will be able to induce anergy in T cells. The objective of the use of apoptotic cells is to make the DCs of the present invention specific so that only the T cell response is modulated for antigens contained in apoptotic cells, which have a relevant role in the pathogenesis of Systemic lupus erythematosus. Moreover, it was evaluated during the experiments carried out that apoptotic cells did not induce maturation of DCs, that is, they did not increase their immunogenic capacity because it was never in the use objectives that modulated the immunogenicity of DCs. Modulation of DC function is achieved using Rosiglitazone (RGZ) and Dexamethasone (DEXA). The publication of Carreño et al. "Induction of Tolerogenic Dendritic Cells by NF-κB Blockade and Fcg Receptor Modulation" describes the use of DCs treated with Rosiglitazone and andrographolide for the treatment of murine multiple sclerosis model and it is suggested that it can be used for other autoimmune diseases such as Lupus where it is unknown the antigen Protocols are given to treat animals directly with these drugs and to generate tolerogenic DCs (tolDCs) with Rosiglitazone and Andrographolide. In the present invention, the use of Rosiglitazone and Dexamethasone is proposed together to generate tolerogenic DCs, which does not appear in the publication of Carreño et al. In addition, in this publication it is proposed to treat mice directly with the NF-κB rosiglitazone and andrografolide inhibitor drugs, not with DCs treated with these drugs as is proposed in the present invention. Treatment with treated DCs is done in a murine multiple sclerosis model. The only mention of the possibility of treating SLE with tolerogenic DCs is subtle and never includes the idea of including apoptotic cells to induce anergy in T lymphocytes that recognize antigens that come from these cells. Moreover, it is specifically mentioned that the antigens in Lupus are

    unknown, it is never proposed that it can be used as a source of antigens to apoptotic cells. Even this remains a potential obstacle. International Patent Application Publication WO2012160200 proposes the use of steroids and vitamin D to generate mature DCs with tolerogenic properties. He
    5 culture time with steroids is 4 days and says textual "to get mature DC". These cells are then cultured in an environment of pro-inflammatory cytokines, where mature DCs are identified by the presence of CD14 on the surface and those tolerogenic by the expression of MERTK. In the present invention it is proposed to obtain immature DCs (iDCs) using Dexamethasone (glucocorticoid) and Rosiglitazone (thiazolidinedione), which is not
    10 mentioned in this patent application and that it is not similar to Vitamin D. The method of the present invention is different, since they are cultured with Rosiglitazone and Dexamethasone. Nowhere in this document is it suggested or proposed that DCs should have specificity given by treatment with any specific antigen. Moreover, nowhere is it suggested that in the case of SLE the source of antigens could be the use of cells
    15 apoptotic. The DCs of the present invention are CD14 (-), that is to say they do not express it and the presence of MERTK is not evaluated, so it is a question of different products originated from different methodologies. For the generation of immunotherapy based on DCs, antigenic specificity is relevant, since one of the advantages of this type of therapy is the decrease in
    20 response against own antigens without affecting an effective response against external agents. However, one of the main obstacles that currently complicate the development of immunotherapy in SLE is the lack of knowledge of the specific autoantigen responsible for the development of the disease. Because patients with lupus have a deficiency in the elimination of cellular waste generated in the
    The apoptosis process is that it has been postulated that these would be a source of autoantigens. This is why the present invention proposes the use of apoptotic cells as a source of

    autoantigens to direct the activity of tolerogenic DCs (tolDCs) exclusively towards self-reactive lymphocytes (Figure 1).
    DESCRIPTION OF THE FIGURES Figure 1. Dendritic cell phenotypes. Immature cells, with low immunogenic potential, mature in the presence of pro-inflammatory stimuli such as LPS, acquiring a phenotype with a high capacity to activate T lymphocytes and an increase in the expression of surface markers CD80, CD83, CD86 and CD40. In contrast, when immature cells receive anti-inflammatory stimuli, they can become tolerogenic cells that induce tolerance in T lymphocytes without the potential to become immunogenic. Figure 2. Experimental design for the generation of Immature Dendritic Cells (iDCs) and Tolerogenic (tolDCs) pulsed with apoptotic cells. Figure 3: Generation of apoptotic cells by UV-B radiation. A: Representative plot plot of lymphocytes not exposed to UV-B (baseline) light. B: Treated with Staurosporine (Novex, Carlsbed, CA, USA) (1µM) for 24 hours (control + apoptosis). C: Treated for 50 minutes at 56 ° C (control + of necrosis). D: Exposed for 1.5 hours to UV-B radiation. E: Percent distribution of lymphocytes per quadrant treated with PI and Anex-V-FITC after 1.5 hours of exposure to UV-B radiation from a control individual. Figure 4. Experimental design for the determination of the ability of DCs to endocyt apoptotic cells. Figure 5: Flow cytometric analysis of the incorporation of apoptotic cells by monocyte-derived DCs. A: Histogram of cell autofluorescence and determination of the CD11c + population. B: Histogram for the determination of positive signal for CFSE (FITC +). C: Population CD11c + of a control individual. D: Histogram of FITC + population obtained from a CD11c + population in cells of a control individual.

    Figure 6: Confocal microscopy of endocytosis of apoptotic cells by DCs derived from monocytes of a patient with SLE in the presence of autologous apoptotic cells. In the left panel of the figure, DCs marked with BODIPY TR Ceramide staining are observed that binds to the Golgi apparatus of living cells. In the center panel, it
    5 appreciate apoptotic cells stained with CFSE. In the right panel, the overlap of the previous images showing the endocytosis of apoptotic cells by DCs (white arrows) is observed. Figure 7: Expression of CD40, CD80, CD83, CD86 and HLA-DR surface markers in human dendritic cells derived from lipopolysaccharide-treated monocytes (LPS)
    10 of S. Typhimurium (1 µg / ml) and co-cultured with autologous apoptotic cells (12.5 µg / ml DNA content, generated by UV-B radiation) in the presence of RGZ (10 µM) and RGZ + Dexa (1 µM) (n = 14 SLE patients). The asterisk indicates * P <0.05 (Friedman Test) when comparing stimulation with LPS and DCs treated with RGZ, DEXA, co-cultured with apoptotic cells and challenged with LPS. The line represents the value that corresponds to the DCs
    15 treated with vehicle. Apocell: apoptotic cells; DEXA: dexamethasone; RGZ: rosiglitazone. Figure 8: IL-6, and IL-12p70 cytokine secretion profile in dendritic cell culture supernatants of patients with SLE derived from monocytes treated with lipopolysaccharide (LPS) of S. Typhimurium (1 µg / ml) and co-cultured with apoptotic cells
    20 autologous (12.5 µg / ml DNA content, generated by UV-B radiation) in the presence of RGZ (10 µM) and DEXA (1 µM). The figure shows a significant decrease in the secretion of IL-6 and IL-12p70 using both treatments. IL-6 is expressed as a “fold increase” with respect to the untreated condition. * P <0.05 Wilcoxon Test. Figure 9: Cell viability test with XTT in the DCs of patients with SLE. The figure
    25 shows that the cell viability of DCs is not altered in the presence of treatment with immunosuppressive drugs (n = 6) when compared to untreated DCs that only

    They receive the vehicle (VEH). Apoptotic cells (Apocell) generated by irradiation with UV-B light were used as a negative viability control. Figure 10: Determination of the activation of peripheral blood lymphocytes (PBL) with markers CD69 and CD71, after being treated with supernatants of DCs cultured in the laboratory for 24 hrs. The figure shows the percentage of CD69 + and CD71 + cells for each condition. n = 1 healthy control. Positive control: cells treated for 24 hrs with Concanavalin A (Con-A, 10 µL / ml); SN: supernatant; Apocell: Apoptotic cells. Figure 11. Experimental design of Mixed Lymphocyte Reaction assay. iDC corresponds to immature dendritic cells, mDC corresponds to mature dendritic cells, and tolDC corresponds to tolerogenic dendritic cells. Figure 12. Functional test of tolDC in mixed lymphocyte reaction with allogeneic T lymphocytes. The expression of CD25 (Figure A) and CD71 (Figure B), and the dilution of the CFSE probe by proliferation, of CD4 + T cells on the fifth day of co-culture with tolDCs was quantified. The graphs show the percentages of CD4 + CD25 +, and CD4 + CD71 + T lymphocytes on the fifth day of co-culture (n = 1). αCD3 + αCD28 is the positive activation control of T lymphocytes. R + D: dexamethasone and rosiglitazone. Figure 13. Functional test of tolDC in mixed lymphocyte reaction with allogeneic T lymphocytes. The expression of CD71 (Figure A), and the dilution of the CFSE probe by proliferation (Figure B), of CD4 + T cells on the fifth day of co-culture with tolDCs was quantified. The graphs show the percentages of CD4 + CD71 + T cells, which proliferated (CD4 + CFSElow) on the fifth day of co-culture. (n = 1). UT: untreated. R / D: dexamethasone and rosiglitazone.
    SUMMARY OF THE INVENTION The present invention consists of tolerogenic dendritic cells (tolDCs) with specific antigens that re-establish the tolerance of the immune system to own organs, a method of producing said tolDCs with specific antigens; use of said tolDCs with antigens

    specific in the production of a therapy for treatment of Lupus Erythematosus
    Systemic (SLE). DESCRIPTION OF THE INVENTION The present invention considers three main aspects. In a first aspect, the
    The present invention corresponds to a method for producing tolerogenic dendritic cells (tolDCs) with specific antigens. In a second aspect, the present invention corresponds to tolerogenic dendritic cells (tolDCs) with specific antigens that restore the tolerance of the immune system to own organs, when administered to a patient in need. Finally, a third aspect of the present invention
    10 corresponds to the use of tolerogenic dendritic cells (tolDCs) with specific antigens in the production of a therapy for the treatment of Systemic Lupus Erythematosus (SLE). An embodiment of the first aspect of the invention corresponds to a method for producing tolerogenic dendritic cells (tolDCs) with specific antigens. In a particular embodiment, the method for producing tolerogenic dendritic cells (tolDCs) with antigens
    15 specific includes the following steps:
    (to) culturing in vitro dendritic cell precursors in animal serum free medium, using cytokines, IL-4 and GM-CSF, to differentiate them into dendritic cells;
    (b) produce apoptotic cells;
    (C) culturing the dendritic cells obtained in step (a) in the presence of compounds with anti-inflammatory activity;
    (d) co-culturing the dendritic cells of step (c) with the apoptotic cells of step (b), so as to promote endocytosis of apoptotic cells by dendritic cells;
    (and) determine through the identification by means of phenotypic evaluation the obtaining of tolerogenic dendritic cells (tolDCs) with specific antigens.

    In a particular embodiment, the dendritic cell precursors of step a) are selected from monocytes, bone marrow progenitors or directly from peripheral blood or umbilical cord blood. In another particular embodiment, differentiation is carried out by culturing the IL-4 and GM-CSF precursors and cytokines under conditions between 30 and 45 ° C, and between 1 and 10% CO2. More specifically at 37 ° C and 5% CO2. In another embodiment, the stage b) apoptotic cells are produced by exposing cells to an apoptotic stimulus selected from ultraviolet type B radiation (UV-B), presence of chemical substances (staurosporine, methotrexate), activation of specific receptors (Fas interaction -Fas ligand) or inhibition of mitochondrial electron transport (heptachlor, rotenone). In another embodiment, the cells from which apoptotic cells come from correspond to blood cells, muscle cells, epidermal cells, epithelial cells, stem cells or human cell lines. In a particular embodiment, the blood cells are peripheral blood lymphocytes, platelets, neutrophils or monocytes. In a more specific embodiment, the blood cells are peripheral blood lymphocytes. In another embodiment, the culture of dendritic cells in the presence of compounds with anti-inflammatory activity of step c) is carried out for a period of between 5 and 48 hours. In a more specific embodiment, compounds with anti-inflammatory activity are selected from rosiglitazone (RZG) and dexamethasone (DEXA) or a combination thereof. In a more specific embodiment, dendritic cells are cultured in the presence of between 5 and 30 µM of RGZ, in the presence of between 0.5 and 5 µM of DEXA. In a specific embodiment, co-culture of the dendritic cells of stage c) with apoptotic cells of stage b) is performed considering an amount of apoptotic cells, expressed as DNA content, between 5 and 20 µg / ml. Medium for animal serum free is used for this, such as AIM-V (GIBCO® AIM V® Medium Grand Island, NY, USA).

    In another embodiment, co-culture of the dendritic cells with apoptotic cells is carried out for a period of between 5 and 48 hours. In another embodiment of the invention, the identification of tolDCs of step e) is carried out by evaluating: (i) the production of the cytokines IL-6, and IL-12p70 that should be decreased with respect to mature immunogenic DCs; and (ii) absence or reduced expression of surface markers compared to immunogenic mature DCs, where the surface markers are selected from CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof. In a more specific embodiment, the evaluation of the production of IL-6, and IL-12p70 and the expression of surface markers CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof is carried out through the Selected technique of ELISA, flow cytometry, Western Blot and also at the level of transcription or messenger RNA by RT-PCR. A second aspect of the invention corresponds to tolerogenic dendritic cells (tolDCs) with specific antigens obtained from the method described above. In a specific embodiment, the specific antigens are autoantigens, and not because they come from a patient, but in autoimmune diseases the immune response is against a proper element, therefore the antigen is autoantigen. In a specific embodiment of the invention, dendritic cells come from: monocytes, bone marrow progenitors or directly from peripheral blood or umbilical cord blood. In another embodiment, specific antigens come from apoptotic cells. In a more specific embodiment, apoptotic cells come from cells that have undergone an apoptotic stimulus. In a specific embodiment, the apoptotic stimulus to which the cells are subjected is selected from ultraviolet radiation type B (UV-B), presence of chemical substances (staurosporine, methotrexate), activation of specific receptors (Fas-Fas ligand interaction) or inhibition of mitochondrial electron transport (heptachlor, rotenone). More specifically, the stimulus used is ultraviolet radiation type B (UV-B).

    In another embodiment, the cells from which apoptotic cells come from correspond to blood cells, muscle cells, epidermal cells, epithelial cells, stem cells or human cell lines. In a particular embodiment, the blood cells are peripheral blood lymphocytes, platelets, neutrophils or monocytes.
    In a specific embodiment, the tolerogenic dendritic cells (tolDCs) of the invention are identified by phenotypic evaluation. In a more specific embodiment, the identification of tolDCs is carried out by evaluating: (i) the production of cytokines IL-6, and IL12p70 that should be decreased with respect to mature immunogenic DCs; and (ii) absence
    or reduced expression of surface markers compared to mature DCs
    10 immunogenic, where the surface markers are selected from CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof. In a more specific embodiment, the evaluation of the production of IL-6, and IL-12p70 and the expression of surface markers CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof is carried out through the Selected ELISA technique, flow cytometry, Western Blot and also at the level of
    15 transcription or messenger RNA by RT-PCR. In a third aspect of the invention, the use of tolerogenic dendritic cells (tolDCs) with specific antigens in the production of a therapy for the treatment of Systemic Lupus Erythematosus (SLE) is described. In a specific embodiment, the invention describes the use of dendritic cells.
    20 tolerogenic (tolDCs) with specific antigens, which serves in the preparation of a drug useful for the treatment of Systemic Lupus Erythematosus (SLE).
    APPLICATION EXAMPLES
    Example 1: Obtaining specific DC for relevant antigens in SLE
    25 Induction of apoptosis by ultraviolet radiation type B (UV-B) in peripheral blood lymphocytes

    At this stage, different exposure times to UV-B radiation were evaluated to obtain apoptotic cells from lymphocytes from peripheral blood from healthy individuals initially and then from patients with SLE. To corroborate the induction of apoptosis, propidium iodide (PI) and Annexin V (Annex) staining was used and the samples were analyzed by flow cytometry. Said cells in apoptosis state are subsequently used to pulse DCs in order to provide autoantigens (Figure 2). Figure 3 shows that an exposure time of 1.5 hrs to UV-B radiation is appropriate to induce apoptosis (PI + Annex +) in these cells.
    Evaluation of the ability of the DCs of patients with SLE to endocyt apoptotic cells.
    Subsequently, it was evaluated whether the DCs generated in the previous test have the ability to endocyte apoptotic cells to process the antigens and then present them to self-reactive T lymphocytes. Apoptotic cells generated by exposure to UV-B radiation were labeled with carboxyfluorescein succinimidyl ester (CFSE) cell staining (Invitrogen, Carlsbad, CA, USA) to be detected with flow cytometry. DCs were incubated for 24 hrs. with CFSE-labeled apoptotic cells. In this experiment, the CD11c + population was selected that allows identifying DCs. The detection of CFSE in the population of CD11c + cells (39.5%) is indicative of phagocytosis of apoptotic cells by DCs (Figures 4 and 5). Briefly, apoptotic cells generated by UV-B radiation were labeled with CFSE and co-cultured for 24 hrs with DCs. The DCs were labeled with BODIPY-TR Ceramide vital staining (Life Technologies) and as seen in Figure 6, the image composition shows that the DCs are capable of endocytizing apoptotic cells both in a control individual (panel B) and in an SLE patients (panel C).
    Effect of co-culture of apoptotic cells and DCs on immunophenotype of DCs.

    It is widely reported that DNA molecules play a role as an autoantigen in patients with lupus and that they are exposed to the extracellular environment during apoptosis associated with proteins such as histones in the form of nucleosomes (1). This is why DNA determination is a way to quantify apoptotic cells,
    5 methodology that has been previously used by other researchers (2). For the results shown in the present invention, a total of 14 patients with SLE were included whose clinical characteristics are detailed in Table 1, from which DCs were generated and on the sixth day they were pulsed with apoptotic cells (12, 5 µg / ml of DNA content) for 24 hours prior to analysis. The state of maturation of the DCs
    The resulting results were analyzed by flow cytometry using anti-CD40, CD80, CD83, CD86 and HLA-DR conjugated antibodies (Figure 7).
    Table 1. Clinical characteristics of patients with SLE for the study of the generation of DC and studies of co-culture with sapoptotic cells. For each patient included in the studies, the degree of activity of the disease is determined through the SLEDAI index, the highest score, the highest activity, an SLEDAI> 6 being defined as an active disease. The average age and criteria for SLE that it meets are also included. each patient Art. Arthritis; Imm. = immunological (presence of anti-DNA, anti-Sm or anti-cardiolipin antibodies); SN: nervous system involvement (seizures or psychosis); Hem. = hematological compromise; Sero = serositis; MC: mucocutaneous; ANA: Anti-nuclear antibodies.

    Effect of RGZ and DEXA on DC pulsed with apoptotic cells.
    Monocyte-derived DCs were obtained from patients with SLE and control individuals and were treated for 24 hours with RGZ (10 µM) and DEXA (1 µM) and then co-cultured with apoptotic cells (12.5 µg / ml DNA content ) for 24 hours
    5 and the expression of markers CD40, CD80, CD83, CD86 and HLA-DR was evaluated in a total of 14 SLE patients (Table 1). In Figure 7, it is observed that DC in the presence of apoptotic cells do not show variation in the expression of any of the maturation markers studied, while treatment with the drugs RGZ + DEXA decreases significantly (p <0.05,
    10 Friedman Test) the expression of the maturation markers, CD80, CD83 and CD86 in DCs of patients with SLE co-cultured with apoptotic cells compared to the cells treated with S. typhimurium LPS, which demonstrates the effectiveness of treatment with Immunosuppressive drugs in changing the phenotype of DCs.
    15 Determination of the activation of DCs: Secretion of proinflammatory and anti-inflammatory cytokines.
    With the purpose of corroborating the induction of a tolerogenic state in the DCs and having more elements that allow as complete a characterization as possible and a functional approach, the production of some cytokines was quantified
    20 relevant in the co-cultures supernatants of the previous experiments, such as: interleukin 6 (IL-6) and interleukin 12p70 (IL-12p70), secreted to the extracellular medium by tolerogenic dendritic cells, by means of the technique of ELISA (enzyme-linked immunosorbent assay). As seen in Figure 8, treatment with RGZ + DEXA induces a decrease
    25 significant (p <0.05) in the secretion of IL-6, a multifunctional cytokine with clearly defined properties in the regulation of inflammatory conditions (3) and that during the antigen presentation plays a fundamental role in the activation and differentiation of

    CD4 + T lymphocytes to any of the effector phenotypes (4), compared to the LPS treated DCs of S. typhimurium. In the same figure there is also a decrease in the secretion of IL-12p70, this cytokine plays an important role in the enhancement of the cytotoxic activity of Natural Killers (NK) cells as well as in the cytotoxic activity
    5 of CD8 + T cells and the development of a pro-inflammatory phenotype in CD4 + T cells. Table 2 summarizes the results obtained and shows that the tolDC product (tolerogenic DCs treated with RGZ and DEXA and pulsed with apoptotic cells) shows a decrease in most of the relevant surface markers associated with maturation and an immunogenic phenotype when challenged with LPS and a decrease in
    10 production of pro-inflammatory cytokines compared to DC not treated with RGZ and DEXA.
    MarkersPhenotypic Cytokines
    CD40 CD80 CD83 CD86 HLA-DR IL-6 IL-12p70
    Immunogenic DC (DCs + LPS) ↑ = ↑ == ↑↑TolDCs (DCs + R + D + Apocell + LPS) = ↓↓↓ = ↓↓
    Table 2. Summary of the phenotype observed in mature DCs and tolDCs. fifteen
    Studies of toxicity and cell viability of therapy for SLE with autologous tolerogenic DCs.
    In order to evaluate the effects of drugs on cell viability and metabolism, the XTT viability test, a colorimetric test that estimates the activity, was carried out
    20 metabolic cell. Its rationale consists in the reduction of the tetrazolium XTT salt, which is transformed into formazan by the activity of mitochondrial dehydrogenase enzymes of metabolically active cells, generating as a product a colored compound, which is generated only in viable cells and whose quantity produced is proportional to the number of viable cells in the sample.

    As seen in Figure 9, drug treatment does not alter the percentage of cell viability of the DCs of patients with SLE. Following the same previous line, an experiment was carried out to evaluate if the generated DCs secrete any potentially toxic substance to the extracellular environment that
    5 could affect the viability of the body cells that will receive the therapy. To perform this test, propidium iodide (PI) was used in conjunction with annexin V (Annex V) which were used to label peripheral blood lymphocytes (PBL) of a control individual that were previously treated with the supernatants of DCs cultured in the conditions of our protocol using immunomodulatory drugs, but without being
    10 submitted to the challenge with LPS and that came from another control individual, for a time of 24 hrs. On the other hand, it was determined whether these supernatants of the generated DCs were capable of producing any activation response in the lymphocytes and for this purpose the CD 69 markers (early activation marker) were determined in the PBL lymphocytes treated and
    15 CD 71 (late activation marker), which are expressed against activation stimuli in lymphoid cell lines. In Figure 10, it is observed that the CD71 marker does not vary its expression when treated with the supernatant of DCs treated with the vehicle nor with the supernatant of DCs treated with drugs and apoptotic cells. With respect to the CD69 marker, it
    20 observes a slight increase in the percentage of activated cells, but which is far from the levels of activation of the positive control corresponding to PBL stimulated with concanavalin A (protein with the ability to induce mitogenic activity in T lymphocytes and increase product synthesis cellular) and that is similar to the results obtained with supernatants of vehicle treated DCs.
    25 From these last two results, it was concluded that DC supernatants do not alter the viability of PBLs, nor do they generate an activation response in these cells.

    Example 2: Functional evaluation of the tolerogenic capacity of DCs
    In the previous example, the qualities and stability of the immunophenotype of tolDCs were characterized as a measure of assessing their potential as therapy and having a functional approach when studying cytokine secretion. To determine your success in
    In a pre-clinical phase, in vitro functional analyzes are required that aim to assess the modulating capacity of the tolDCs generated on CD4 + T lymphocytes. An allogeneic Mixed Lymphocyte Reaction (MLR) assay was then performed using DCs and CD4 + T cells from different healthy individuals (Figures 11 and 12).
    10 TolDCs of an individual and T cells of a different individual previously stained with CFSE were cultured in 200 µL of RPMI 1640 medium + 10% FBS (1: 5 ratio) for 5 days. As a control, co-cultured T lymphocytes with immature DCs (iDCs), mature DCs were used
    or without DCs until the end point of the experiment. The activation of T lymphocytes was evaluated on the fifth day by flow cytometry through the expression CD25 and CD71 (Figure 12).
    The expression of CD25 and CD71 (T lymphocyte activation markers) of T cells cultured in the presence of tolDCs induced by each drug or both simultaneously, is lower than that observed for cells co-cultured with immature DCs. The results demonstrate the tolerogenic function of DCs induced by treatment with NF-κB inhibitor drugs.
    The same trial was also performed by taking tolDCs from a patient with SLE and T cells from a healthy individual previously stained with CFSE and were cultured in 200 µL of RPMI 1640 medium + 10% FBS (ratio 1: 5) for 5 days. As a control, T lymphocytes co-cultured with immature DCs, mature DCs or without DCs were used until the end point of the experiment. Proliferation and activation of T lymphocytes was evaluated on the fifth day by cytometry of
    Flow through measurement of CFSE dilution and CD71 expression (Figure 13). The expression of CD71 (T lymphocyte activation marker) of T cells cultured in the presence of tolDCs induced by each drug or both simultaneously, is

    lower than that observed for cells co-cultured with immature DCs. Further,it was observed that T lymphocytes proliferate less in the presence of tolDCs than of DCsmatureExample 3: Protocol for the generation of tolDCs in Systemic Lupus Erythematosus.
    5 A. Isolation of peripheral blood or leukocyte layer should not be greater than 8hours and supplemented with Heparin
    1. Blood was distributed in 50 ml conical tubes and diluted using 1X PBS to reach 35 ml of total volume.
    2. 15 ml of Ficoll-Paque lymphocyte separation medium was added to the bottom of 10 an empty 50 ml conical tube.
    3. The diluted blood was carefully transferred to each 50 ml tube containing the Ficoll-Paque medium.
    Four. It was centrifuged at 1000xg for 25 minutes at 20 ° C.
    5. The upper layer (serum) is aspirated leaving the mononuclear cell layer unchanged at the interface.
    6. The mononuclear cell layer was carefully transferred to a new 50 ml conical tube.
    7. The 50 ml conical tube containing the mononuclear cell layer was filled with
    1X PBS and centrifuged at 300xg at 20 ° C for 10 minutes. The supernatant was carefully removed and discarded.
    8. The pellet was resuspended in 5 ml of RBC lysis buffer (ACK 1X) for 5 minutes at room temperature.
    9. The 50 ml conical tube was filled with 1X PBS and mixed gently.
    10. It was centrifuged at 300xg for 10 minutes and the supernatant was discarded. 25 11. Steps 7, 8 and 9 were repeated once.
    12. The pellet was resuspended in 5 ml of preheated AIM-V medium. Cell count was performed.

    13. The cells were planted in 6 6-well 10x106 PBMCs plates in 1 mL of AIM-V medium.
    14. It was incubated for 2 hours at 37 ° C, 5% CO2 and continued in section B. 5 B. Obtaining peripheral blood lymphocytes and differentiation to DC.
    1. The supernatant was carefully removed to take peripheral blood lymphocytes (PBL) without touching the bottom of the tube. (PBL was stored until generating apoptotic cells). It was washed 3 times with 1 ml of preheated 1X PBS at 37 ° C.
    2. 1.5 mL of preheated AIM-V medium containing IL-4 (1000 IU / mL) and 10 GM-CSF (1000 IU / mL) (Day 1) was added. It was incubated at 37 ° C, 5% CO2.
    3. Fresh cytokines (IL-4 and GM-CSF) were added on day 3 and 5, to a final concentration (1000 IU / mL) of the culture without changing the medium. C. Generation of apoptotic cells
    15 1. The non-adherent fraction (PBL) was transferred to a 50 mL conical tube and the tube was filled with 1X PBS.
    2. It was centrifuged at 300xg for 6 minutes.
    3. It was re-suspended in 5 mL of the AIM-V medium and planted in a T-25 culture flask at 37 ° C with 5% CO2. The AIM-V medium was changed every day.
    20 4. A UV lamp was mounted inside the biosafety cabinet and the lamp was preheated for 10 minutes.
    5. PBL was carefully transferred to a sterile 60x15 ml plate.
    6. The lymphocytes were irradiated for 1.5 hours, at 2.0 mW / cm2. Proceed with section D.
    25 D. Co-culture of DCs and apoptotic cells (Day 7)


    one. Apoptotic cells were homogenized and the final volume determined after UV treatment. The concentration of AND was determined using 400 µL of the apoptotic cell preparation.
    2. The cells were transferred to a conical tube and centrifuged at 500xg for 10 minutes.
    5 3. The supernatant was carefully discarded and the pellet was resuspended to afinal concentration of 1 µg / mL of DNA.
    4. 18.75 µL of the apoptotic cell preparation was added to the DC culture medium prepared in section A. 10 E. Induction of tolerogenic DC (Day 6)
    1. Rosiglitazone was dissolved in DMSO to prepare a stock solution (100 µL DMSO / 1.79 mg rosiglitazone). De diluted 5 µL of stock solution with 495 µL of 1X PBS to prepare the working solution. 30 µL of the working solution was added to the DC cell culture (final concentration = 10 µM).
    Dexamethasone was dissolved in DMSO to prepare a stock solution (100 µL DMSO / 0.2 mg dexamethasone). 5 µL of stock solution was diluted with 20 µL of 1X PBS to prepare the working solution. 1.5 µL of the working solution was added to the DC cell culture (final concentration = 1 µM). 20 REFERENCES
    (one) Rosen A, Casciola-Rosen L. Autoantigens in systemic autoimmunity: critical partner in pathogenesis. Journal of internal medicine. Jun 2009; 265 (6): 625-631.
    (2) Fehr EM, Spoerl S, Heyder P, et al. Apoptotic-cell-derived membrane vesicles induce
    25 an alternative maturation of human dendritic cells which is disturbed in SLE. J Autoimmun. Feb 2013; 40: 86-95.

    (3) Wolf J, Rose-John S, Garbers C. Interleukin-6 and its receptors: a highly regulated and dynamic system. Cytokine 2014 Nov; 70 (1): 11-20. doi: 10.1016 / j.cyto. 2014.05.024. Epub 2014 Jun 28. Review. PubMed PMID: 24986424.
    (4) Zhu J, Paul W. CD4 T cells: Fates, functions and faults. Blood, 2008; 112 (5): 15575 1569.
    (5) Nagy ZS, Czimmerer Z, Szanto A, Nagy L. Pro-inflammatory cytokines negatively regulate PPARγ mediated gene expression in both human and murine macrophages via multiple mechanisms. Immunobiology 2013 Nov; 218 (11): 1336-44. doi: 10.1016 / j.imbio. 2013.06.011. Epub 2013 Jul 1. PubMed PMID:
    10 23870825.
    (6) Hontelez S, Karthaus N, Looman MW, Ansems M, Adema GJ. DC-SCRIPT regulates glucocorticoid receptor function and expression of its target GILZ in dendritic cells. J Immunol. 2013 Apr 1; 190 (7): 3172-9. doi: 10.4049 / jimmunol. 1201776. Epub 2013 Feb 25. PubMed PMID: 23440419.
    权利要求:
    Claims (19)
    [1]
    1. A method for producing tolerogenic dendritic cells (tolDCs) with specific antigens, CHARACTERIZED because it comprises the following steps:
    5 (a) culturing dendritic cell precursors in animal serum free medium, using cytokines, IL-4 and GM-CSF, to differentiate them into dendritic cells;
    (b) produce apoptotic cells;
    (C) culturing the dendritic cells obtained in step (a) in the presence of compounds with anti-inflammatory activity;
    10 (d) co-culturing the dendritic cells of step (c) with the apoptotic cells of step (b), so as to promote endocytosis of apoptotic cells by dendritic cells;
    (e) determine through the identification by means of phenotypic evaluation the obtaining of tolerogenic dendritic cells (tolDCs) with specific antigens.
    The method according to claim 1, CHARACTERIZED in that the dendritic cell precursors of step a) are selected from monocytes, bone marrow progenitors or directly from peripheral blood or umbilical cord blood.
    [3]
    3. The method according to claim 1, CHARACTERIZED because the differentiation
    It is carried out by cultivating the precursors and cytokines IL-4 and GM-CSF in conditions of 20 between 30 and 45 ° C, and between 1 and 10% of CO2.
    [4]
    4. The method according to claim 1, CHARACTERIZED in that the apoptotic cells of step c) are produced by exposing cells to an apoptotic stimulus selected from ultraviolet radiation type B (UV-B); presence of selected chemical substances of staurosporine, methotrexate; activation of specific receptors such as

    Fas-Fas interaction ligand or inhibition of mitochondrial electron transport with heptachlor or rotenone.
    [5]
    5. The method according to claim 1, CHARACTERIZED in that the cells from which the apoptotic cells come from correspond to blood cells,
    5 muscle cells, epidermal cells, epithelial cells, stem cells or human cell lines.
    [6]
    6. The method according to claim 5, CHARACTERIZED because the blood cells are peripheral blood lymphocytes, platelets, neutrophils or monocytes.
    [7]
    7. The method according to claim 1, CHARACTERIZED because the cultivation of the
    10 dendritic cells in the presence of compounds with anti-inflammatory activity of stage d) are performed for a period of between 5 and 48 hours.
    [8]
    8. The method according to claim 7, CHARACTERIZED in that the compounds with anti-inflammatory activity are selected from rosiglitazone (RZG) and dexamethasone (DEXA) or a combination thereof.
    The method according to claim 8, CHARACTERIZED in that the dendritic cells are cultured in the presence of between 5 and 30 µM of rosiglitazone, and in the presence of between 0.5 and 5 µM of dexamethasone.
    [10]
    10. The method according to claim 1, CHARACTERIZED in that the co-culture of the dendritic cells of stage c) with apoptotic cells of stage b) is performed
    20 considering an amount of apoptotic cells, expressed as DNA content, between 5 and 20 µg / ml.
    [11]
    11. The method according to claim 10, CHARACTERIZED because it is co-cultivated in animal serum free medium.


    [12]
    12. The method according to claim 1, CHARACTERIZED in that the co-culture of the dendritic cells with the apoptotic cells is carried out for a period of between 5 and 48 hours.
    [13]
    13. The method according to claim 1, CHARACTERIZED because in step e),
    5 the identification of tolDCs is carried out by evaluating: (i) the production of the cytokines IL-6, and IL12p70 that must be decreased with respect to mature immunogenic DCs; and (ii) absence
    or reduced expression of surface markers compared to immunogenic mature DCs, where the surface markers are selected from CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof.
    The method according to claim 13, CHARACTERIZED in that the evaluation of the production of IL-6, and IL-12p70 and the expression of surface markers CD40, CD80, CD83, CD86, HLA-DR, or combinations of them is performed through the selected ELISA technique, flow cytometry, Western Blot and also at the level of transcription or messenger RNA by RT-PCR.
    15. Tolerogenic dendritic cells (tolDCs) with specific antigens, CHARACTERIZED because they are obtained from the method of claim 1.
    [16]
    16. Tolerogenic dendritic cells with specific antigens according to claim 15, CHARACTERIZED because they come from: monocytes, bone marrow progenitors or directly from peripheral blood or umbilical cord blood.
    The tolerogenic dendritic cells with specific antigens according to claim 15, CHARACTERIZED because the specific antigens are autoantigens.
    [18]
    18. Tolerogenic dendritic cells with specific antigens according to claim 15, CHARACTERIZED because the specific antigens come from apoptotic cells.


    [19]
    19. Dendritic cells tolerogenic with specific antigens according to claim 15, CHARACTERIZED because apoptotic cells come from cells that have undergone apoptotic stimulation.
    [20]
    twenty. Tolerogenic dendritic cells with specific antigens according to the
    5 claim 19, CHARACTERIZED in that the apoptotic stimulus to which the cells are subjected is selected from ultraviolet radiation type B (UV-B); presence of selected chemical substances of staurosporine, methotrexate; activation of specific receptors such as Fas-Fas ligand interaction or inhibition of mitochondrial electron transport with heptachlor or rotenone.
    21. The tolerogenic dendritic cells with specific antigens according to claim 19, CHARACTERIZED because from which the apoptotic cells come from correspond to blood cells, muscle cells, epidermal cells, epithelial cells, stem cells or human cell lines.
    [22]
    22. Tolerogenic dendritic cells with specific antigens according to the
    Claim 21, CHARACTERIZED because the blood cells are peripheral blood lymphocytes, platelets, neutrophils or monocytes.
    [23]
    23. Tolerogenic dendritic cells with specific antigens according to claim 15, CHARACTERIZED because they are identified by phenotypic evaluation.
    24. The tolerogenic dendritic cells with specific antigens according to claim 23, CHARACTERIZED in that the identification of tolDCs is carried out by evaluating: (i) the production of the cytokines IL-6, and IL-12p70 which must be decreased with respect Immunogenic mature DCs; and (ii) absence or reduced expression of surface markers compared to mature immunogenic DCs, where the

    Surface markers are selected from CD40, CD80, CD83, CD86, HLA-DR, or combinations of them.
    [25]
    25. Tolerogenic dendritic cells with specific antigens according to claim 24, CHARACTERIZED because the evaluation of the production of IL-6, and IL
    5 12p70 and the expression of surface markers CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof is performed through the selected technique of ELISA, flow cytometry, Western Blot and also at the level of transcription or Messenger RNA by RT-PCR.
    [26]
    26. Use of tolerogenic dendritic cells (tolDCs) with specific antigens,
    CHARACTERIZED because it serves in the preparation of a drug useful for the treatment of Systemic Lupus Erythematosus (SLE).

    Fig. 1
    Fig 2

    Fig. 3

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    Fig. 6

    Fig. 7

    Fig. 8
    Fig. 9

    Fig. 10
    Fig. 11

     (A) (B) 
    Fig. 12
     (A) (B)
    Fig. 13
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    同族专利:
    公开号 | 公开日
    SG11201610200RA|2017-01-27|
    JP2017518080A|2017-07-06|
    GB201620621D0|2017-01-18|
    MX2016016023A|2017-03-28|
    CL2015001539A1|2015-09-21|
    AR100657A1|2016-10-26|
    US20170196950A1|2017-07-13|
    CA2951222A1|2015-12-10|
    WO2015186105A2|2015-12-10|
    GB2541600A|2017-02-22|
    WO2015186105A3|2016-01-28|
    ES2609068B1|2018-05-04|
    ES2609068R1|2017-06-29|
    CN106471115A|2017-03-01|
    BR112016028552A2|2017-08-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP1056834A2|1998-02-20|2000-12-06|The Rockefeller University|Apoptotic cell-mediated antigen presentation to dendritic cells|
    WO2013036293A1|2011-09-06|2013-03-14|Selecta Biosciences, Inc.|Dendritic cell subsets for generating induced tolerogenic dendritic cells and related compositions and methods|CN109310746A|2016-06-24|2019-02-05|麦克马斯特大学|Adoptive cellular transfer and oncolytic virus combination treatment|
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    优先权:
    申请号 | 申请日 | 专利标题
    US201462008319P| true| 2014-06-05|2014-06-05|
    US62/008,319|2014-06-05|
    PCT/IB2015/054267|WO2015186105A2|2014-06-05|2015-06-05|Method for producing autologous tolerogenic dendritic cellswith specific antigens and use thereof in the production of a drug suitable for the treatment of systemic lupus erythematosus |
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